The Screening Of The Broad-spectrum Antagonistic Bacteria And The Purification Of Their Antifungal Substances

1. The screening of the antagonistic bacteriaAntagonistic bacteria I7 and M22BH3 are obtained by screening on plate , whose inhibition activity against 16 phytopathogenic fungi are strongest among 501 bacteria isolated from soil samples. Antagonistic bacterium I7 is isolated from soil sample in eggplant ground of annin area, Lanzhou city, Gansu province, and antagonistic bacterium M22BH3 is isolated from soil sample in lily ground of fanjiaping , Lanzhou city, Gansu province. Antagonistic bacteria I7 and M22BH3 both have strong inhibition effect against 16 phytopathogenic fungi, whose pathogen inhibiting zone diameters even reach to the 37.2 mm and 36.9 mm respectively. Their cell-free filtrates also have strong inhibition effect against 16 phytopathogenic fungi, whose pathogen inhibiting rate of their original cell-free filtrates reach to 82.6% and 83.3 % respectively.2. The phylogenetic analysis of antagonistic bacteria I7 and M22BH3Through a series studies of their Characteristics, including the appearance observation, environment factor influence , growing characteristic , physiological and biochemical determination, sequence analysis of 16S rRNA and (G+C)mol% measurement, the determination show that antagonistic bacterium I7 is a new strain of Bacillus amyloliquefaciens, who is already conserved in CCTCC with number M206027, named Bacillus amyloliquefaciens I7, and antagonistic bacterium M22BH3 is a new strain of Bacillus subtilis, who is already conserved in CCTCC with number M206028, named Bacillus subtilis M22BH3. Now we have applied the patents on these two antagonistic bacteria in SIPO of the PRC, and the patent numbers respectively are 200610200364X and 2006102003654.3. The purification and property research of the antifungal substances from I7 and M22BH3 The cell-free filtrates of antagonistic bacteria I7 and M22BH3 are proved both having theproperty to resisting strong heat, such as, the cell-free filtrates of antagonistic bacteria I7 and M22BH3 can retain their 75% and 71.4% inhibition activity respectively after heating at 121℃ for 20min, and the property not sensitive to proteinase K and amylopsin. Through a series of purification as (NH₄)₂SO₄ rating saturation, column chromatography on Sephadex G-50 and column chromatography on DEAE- sephadex A-25, the crude antifungal substances are extracted from the cell-free filtrates of antagonistic bacteria I7 and M22BH3 respectively. The massspectrum analysis results show that the the crude antifungal substances both are the mixture of polypeptides and small molecular proteins.